This study reported the production of pH-responsive films based on 8 wt% polyvinyl alcohol solution/0.2 wt% agar solution incorporated with cochineal-loaded starch particles (CSN)  by a casting process. Results revealed that CSN presented obvious color changes over the pH range of 2–12. FTIR, XRD spectra and SEM micrographs presented that the incorporation of CSN formed new hydrogen bonds with a matrix and a tighter network structure. A certain improvement was observed in the color stability, swelling index and functional properties  but water solubility, water vapor permeability and water contact angle of the pH-responsive films were decreased by the addition of CSN. The release of cochineal was a rate-limiting step following the Korsmeyer-Peppas model. The agar/polyvinyl alcohol film containing 6% CSN (PVA/GG-6) exhibited the best sensitivity for ammonia detection and its limit of detection was 35.4 ppm (part per million) for ammonia. The application trials showed that the PVA/GG-6 film presented different color changes for pork freshness. Hence, these pH-responsive films can be used as potential packaging materials for tracking the freshness of protein-rich fresh food in a non-destructive way. As the demand for food safety increases, consumers are paying more attention to the quality of food. Packaging is a useful way of protecting food from the external environment  to guarantee food health and safety . This haA large number of biodegradable natural polymers  have been used for the fixation of pH-sensitive dyes ,9,10. AgCochineal (CL) is a stable and safe natural pigment extracted from dried female insects, such as Dactylopius coccus . These iIn the previous reports, a large number of natural active compounds  have been used for the development of new active food packaging materials. Among the natural antioxidants/antimicrobials that have been used, essential oils (EOs), thyme oil, ZnO and anthocyanins have been extensively studied for use in active food packaging systems ,22,23. MSince cochineal exhibits different colors at different pH values and studies on cochineal-based active smart packaging are not available, pH-responsive films based on polyvinyl alcohol/agar incorporated with cochineal-loaded starch particles were fabricated by the casting method. The effect of different cochineal-loaded starch particle contents on the color stability, water solubility, swelling property, water vapor permeability and water contact angle and functional properties of the films were considered. Additionally, the release kinetics of cochineal from the matrix through different models were researched. To test the application effect of the pH-responsive intelligent packaging film, it was applied to pork for monitoring the freshness changes during refrigeration.6H10O5)n·xH2O, CAS No-9037-22-3) and Diammonium 2, 2’-azino-bis(3-ethylbenzothiazoline-6-sulfonate)  were obtained by Macklin Biochemical Co., Ltd. . Polyvinyl alcohol 1799 , agar ((C12H18O9)n, density: 0.877, CAS No-9002-18-0), dimethyl sulfoxide , glycerol , ammonia hydrogen solution (25% in water) and acetic acid  were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. . S. aureus and E. coli were supplied by the Hunan University of Technology .Fresh pork was obtained from a local market . Cochineal, amylopectin , and amylopectin particles were slowly added at a mass ratio of 1:10. The mixture was stirred for 3 h to obtain a cochineal-loaded starch particles suspension.2 = 0.99958, where x (μg·mL−1) represents the concentration of cochineal and y represents the absorbance of the solution at 280 nm. The EE was obtained according to the equation:C1 and C2 are the free cochineal concentration and total cochineal concentration, respectively.The suspension was centrifuged at 10,000 rpm for 30 min to obtain the composite precipitate. The absorbance of the supernatant was measured using a spectrophotometer  at 496 nm. The free cochineal in the solution can be obtained from the standard curve : y = 25.The particle sizes of the samples were tested according to the method of Teng et al. . All samTo analyze the pH-sensitivity property of cochineal, the 1.0 mg cochineal was dissolved in 50 mL of buffer solution (pH 2–12) to obtain the cochineal solution (0.02 mg/mL). The color information of the cochineal solution was observed and photographed. The absorption wavelength of the cochineal solution was characterized by an ultraviolet-visible spectrometer  from 400 to 700 nm .v/v and stirring for 2 h at 60 °C. Glycerol was added to the mixed solution as a plasticizer (5% w/w mass of the mixed solution). When cooled to 70 °C, the polyvinyl alcohol/agar solution was added to the desired cochineal-loaded starch particles or cochineal powder  and continuously stirred to obtain the film-forming solution. Then, an appropriate amount of acetic acid is added to adjust the pH (2–3) of the film-forming solution. Lastly, 15 g of the solution was uniformly cast onto a glass Petri dish (diameter = 90 mm) and then dried in an oven  at 45 °C for 48 h to obtain all films. The preparation schematic diagram was presented in The pH-responsive films were prepared by a modified method . Polyvin−1. X-ray diffraction of all samples was obtained using an X-ray diffractometer  with the scanning range of 5–90° (2θ) under Ni-filtered Cu-Kα radiation.The microstructure of the film was recorded using SEM . The samples were treated in liquid nitrogen for 10 min to obtain brittle fracture samples before the test. The truncated surface was glued to the conductive adhesive, and the sample was obtained by spraying gold for 45 s. The acceleration voltage during SEM shooting was 3 kV. The FT-IR analysis of the films was captured by an FT-IR spectrometer  from 1000 to 4000 cmE*) of the film was evaluated in accordance with the following formula.L* (91.72), a* (0.61), b* (−0.25) is the color values of the standard whiteboard.The color information of the film was recorded using a colorimeter  . The totm (g) of the sample ..42]. Thetic ring . The disectively . AmylopeL value of the film decreased, while the a and b values increased, indicating that the pH-responsive films became redder and yellower. This was because the more cochineal in the film, the darker the orange color of the film, the L value decreased and the a value and b value increased correspondingly.The color parameters and stability of the films were shown in E*) of all films stored under identical lighting conditions. The storage conditions of the films are critical for their application. As shown in The color stability of the pH-responsive films was characterized by measuring the color difference values (ΔIn 2). The Korsmeyer–Peppas model was used to simulate the release behavior of cochineal from the film at Mt/M0 ≤ 2/3. The high value of R2 (0.96015–0.98674) confirmed that the Korsmeyer–Peppas model could be effectively employed to analyze the release behavior of cochineal. The release index n (0.16089–0.23448) showed that cochineal release followed Fick diffusion [2 > 0.97629) and first-order kinetic equation (R2 > 0.91998), suggesting that the diffusion mechanism of cochineal was a rate-limiting step.To further investigate the release behavior of cochineal in the films, three different typical models were applied to fit and analyze its release behavior  [The growth and antibacterial curves of  E. coli . The resembrane) .+∙) showed that cochineal possessed the ability to scavenge free radicals [+∙ became stable ABTS while cochineal was converted into a semi-quinone free radical stable structure [Spoilage and nutrition loss of food are related to free radicals . Cochineradicals . In thistructure . Since t3 also increased as the concentration of ammonia vapor increased  of 0.98629 (LOD) for ammonia vapor in the PVA/GG-6 film were calculated as 39.4 ppm using the following formula. The results indicated that the PVA/GG-6 film could be used to detect the freshness of aquatic products and meat (35 ppm) in food packaging [S was the standard deviation (0.016401) of the blank group and N was the slope of the fitted curve.The PVA/GG-6 film with the highest gas sensitivity was selected to determine its detection limit for NH 0.98629 C. Hence,10 CFU/g, respectively. As the storage time increased, a decrease in pH (6.85) occurred on the first day, and then gradually increased to 6.26 on the 7th day. This may be due to the production of lactic acid from the anaerobic digestion of glycogen and phosphate production from adenosine triphosphate degradation in pork [10 CFU/g to 18.71 mg/100 g and 6.08 log10 CFU/g, respectively, representing an increase of 510.7% and 137.9%. These exceed the limits of 15 mg/100 g (GB5009.228-2016) and 6 log10 CFU/g (GB 4789.2-2016) according to Chinese standards, thus indicating that pork was not edible after 6 days of storage in the refrigerator [In  in pork . HoweverE* value of the PVA/GG-6 film showed a positive correlation with TVB-N, pH and TVC values with high correlation coefficients. The TVB-N value showed a positive correlation with both pH and TVC values, with a correlation coefficient ranging from 0.81 to 0.98. The results showed that the novel freshness indicator film was feasible for freshness monitoring of refrigerated pork. Consequently, both the application object and the pH-colorant content strongly influence the effectiveness of freshness monitoring using this indicator film.As shown in E* value of the PVA/GG-6 film showed a positive correlation with the TVB-N and TVC values of pork, with a high correlation coefficient. The color change of the PVA/GG-6 film can fully indicate the spoilage of refrigerated pork, enabling consumers to judge the freshness of cold meat products with their naked eyes.As the pH increased from 2 to 12, the cochineal color shifted from orange to red and then to purple. The FIIR spectrum of the pH-responsive films indicated successful immobilization of CSN in the PVA/GG matrix, which can be attributed to newly generated interactions between the polymer matrix and the natural dye. The wettability and swelling properties of the pH-responsive films were influenced by the incorporation of CSN. Due to the poor photostability of cochineal, the photostability of pH-responsive films was significantly enhanced by encapsulation of cochineal with amylopectin particles. The release of cochineal from pH-responsive film followed the Fickian diffusion mechanism, which can be described by both a first-order model and a second-order model. The excellent antioxidant and antibacterial activities of pH-responsive films resulted from the release of cochineal. The Δ


The cryo-EM structure, together with molecular dynamics simulations and mutagenesis studies, revealed the molecular basis of CMKLR1 signaling by elucidating the interactions at the ligand-binding pocket and the agonist-induced conformational changes. Our results are expected to facilitate the development of small molecule CMKLR1 agonists that mimic the action of chemerin9 to promote the resolution of inflammation.Chemokine-like receptor 1 (CMKLR1), also known as chemerin receptor 23 (ChemR23) or chemerin receptor 1, is a chemoattractant G protein–coupled receptor (GPCR) that responds to the adipokine chemerin and is highly expressed in innate immune cells, including macrophages and neutrophils. The signaling pathways of CMKLR1 can lead to both pro- and anti-inflammatory effects depending on the ligands and physiological contexts. To understand the molecular mechanisms of CMKLR1 signaling, we determined a high-resolution cryo-electron microscopy (cryo-EM) structure of the CMKLR1-G Chemokine-like receptor 1 (CMKLR1) responds to the adipokine chemerin and is highly expressed in innate immune cells. A high-resolution structure of CMKLR1 in complex with G protein and a peptide agonist, together with mutagenesis and computational simulations studies, reveals mechanisms of peptide agonist recognition, receptor activation, and G protein-coupling. The double-mutant cycle analysis indicates that Tyr149 of chemerin9 is not exclusively interacting with F2947.31 of CMKLR1 -C9) and the other changing the carboxylate group of Ser157 to carboxamide (C9-NH2) (Table 2). While the carboxamide group in C9-NH2 is likely to be positively charged, [A9]-C9 still maintains the last negatively charged carboxylate group. Compared to [A9]-C9, C9-NH2 caused a more significant decrease in potency, indicating that the negative charge of the C-terminal end of chemerin9 is critical to ligand binding . Moreover, while C9-NH2 showed no activity on the 3 CMKLR1 variants, R1784.64A, R1784.64K, and R1784.64Q, it partially rescued the loss of activity of chemerin9 on the CMKLR1 R2245.42A and R2245.42Q variants . It is possible that the salt bridge between the C-terminal carboxylate of chemerin9 and R2245.42 of CMKLR1 is essential for maintaining the ligand in the right conformation for activating the receptor. For C9-NH2, the carboxamide group at the C-terminus may form polar interactions with other nearby residues in the CMKLR1 R2245.42A and R2245.42Q variants, thereby stabilizing the carboxamide group in the correct position. However, in the wt CMKLR1 or the R2245.42K variant, the same-charge repulsion may cause this group of C9-NH2 to swing away from R224 (wt) or K224 (variant), resulting in the significantly lower activity of C9-NH2 on both receptors.At the C-terminal end of chemerin9, which is bound at the bottom of the binding pocket, the carboxylate group of the last residue Ser157 is located in a highly positively charged environment surrounded by CMKLR1 residues R1782.53, H952.60, M1233.36, Y2766.51, and I3067.43 . Consistently, when Phe156 was mutated to Ala in chemerin9, the resulting peptide ([A8]-C9) . We also examined the impact of mutations at 3 aromatic residues in CMKLR1, F882.53, H952.60, and Y2766.51, on ligand binding. Mutating F882.53 to Ala decreased the potency of chemerin9 by approximately 100-fold, but changing it to Leu or His led to a more significant decrease in agonist potency. These results suggest that neither the aromaticity nor the size of F882.53 is important for the action of chemerin9. Furthermore, there was a gain of potency of [A8]-C9 for the F882.53A and F882.53L variants compared to the wt CMKLR1. Therefore, while F882.53 is important in chemerin9 binding, the role of this residue appears to be complex. In the case of H952.60, mutation to Ala, Leu, or Lys reduced the potency of chemerin9 to different extents, possibly by altering its interaction with Phe156 of chemerin9. For Y2766.51, exchange by Ala completely abolished the activity of chemerin9, likely due to poor cell surface expression of the receptor variant . Interestingly, when mutated it to Leu, the potency of chemerin9 decreased by 10-fold, while mutation to Phe increased the maximum activity of chemerin9 , indicating the importance of the aromaticity of Y2766.51 for the activity of chemerin9. None of the mutations of H952.60 or Y2766.51 rescued the activity of [A8]-C9.Phe156 of chemerin9 plays a crucial role in the interactions with the receptor, forming hydrophobic or π–π interactions with CMKLR1 residues F88i coupling, we performed 5 different sets of MD simulations of the CMKLR1-Gi complex or CMKLR in the absence or presence of chemerin9 (see details in Methods). Each system was subjected to 3 runs of 100-ns MD simulations, resulting in a total of 1.5 microseconds of simulation data. The CMKLR1-Gi complex remained stably associated throughout all simulation runs with or without chemerin9. Noticeably, we observed that CMKLR1 exhibited slightly more conformational fluctuations in the absence of chemerin9, despite being coupled and stabilized by Gi . To investigate the impact of charges of chemerin9 on ligand binding, we conducted simulations of CMKLR1 bound to chemerin9 with both neutral and charged amino- and carboxyl-termini (N-ter and C-ter). Both charged and noncharged chemerin9 remained stably bound in the binding pocket throughout the simulations, indicating that the binding is overall stabilized by a network of interactions rather than specific pairs. Yet, to further evaluate the binding energy of chemerin9, we used the Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) method -C9, and C9-NH2. The amidated C-terminus has an increased influence compared to the Ala change at position 9 in C9. (d) Positions of Y471.39, N1914.77, S2957.32, and L2987.35 of CMKLR1 and (e) signaling of their Ala variants stimulated by C9. All signaling assays were performed by measuring Ca2+ influx. The assays were executed at least 3 times in triplicates. Results are represented as means ± SEM. The underlying data for S4A, S4B and S4E Fig can be found in (TIF)Click here for additional data file.S5 Fig6.51A, H952.60A, and H952.60L are located within the cell, whereby the F882.53A variant is only partially expressed in the membrane.HEK293 cells were transiently transfected with 1,000 ng of receptor variant fused with eYFP with Lipofectamine 2000. Receptor expression was determined by eYFP autofluorescence. Cell nuclei were stained with Hoechst33324 (blue). Scale bar 10 μm. Most receptor variants are expressed in the membrane, but Y276(TIF)Click here for additional data file.S6 Fig(a) RMSD of the receptor alone and the CMKLR1-Gi complex in the simulations of the CMKLR1-Gi complex with (left) and without (right) chemerin9. CMKLR1 deviated from that in the cryo-EM structure within 2.0 ± 0.5 Å, and the entire protein complex (CMKLR1 + chemerin9 + G protein) deviated from that in the cryo-EM structure within 3.5 ± 1.0 Å. In the absence of chemerin9, CMKLR1 showed more fluctuations. The underlying data for S6A Fig can be found in (b) Interfacial salt bridges intermittently formed between CMKLR1 and the Gi complex during the simulations. CMKLR1, chemokine-like receptor 1; cryo-EM, cryo-electron microscopy; MD, molecular dynamics; RMSD, root-mean-square deviation.(TIF)Click here for additional data file.S7 Fig(a and b) The comparison of the overall structure and the chemerin9 binding pocket, respectively. Chemerin9 in our structure and in the published structure is colored orange and yellow, respectively. The structural alignment is based on the receptor.(TIF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S3 Data(XLSX)Click here for additional data file.S4 Data(XLSX)Click here for additional data file.S5 Data(XLSX)Click here for additional data file.S6 Data(XLSX)Click here for additional data file.S1 Raw image(PDF)Click here for additional data file. 7 Jun 2023Dear Dr Zhang, Thank you for submitting your manuscript entitled "Structural basis of CMKLR1 signaling induced by chemerin9" for consideration as a Research Article by PLOS Biology. Please accept my sincere apologies for the long delay in getting back to you as we consulted with an academic editor about your submission. Your manuscript has now been evaluated by the PLOS Biology editorial staff, as well as by an academic editor with relevant expertise, and I am writing to let you know that we would like to send your submission out for external peer review under our scooping protection policy. However, before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.https://www.editorialmanager.com/pbiology) within two working days, i.e. by Jun 09 2023 11:59PM.Once your full submission is complete, your paper will undergo a series of checks in preparation for peer review. After your manuscript has passed the checks it will be sent out for review. To provide the metadata for your submission, please Login to Editorial Manager  corporation #C2354500, based in San Francisco 8 Aug 2023Dear Dr Zhang,Thank you for your patience while your manuscript "Structural basis of CMKLR1 signaling induced by chemerin9" was peer-reviewed at PLOS Biology. Please accept my sincere apologies for the delays that you have experienced during the peer review process. Your manuscript has now been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by three independent reviewers. In light of the reviews, which you will find at the end of this email, we would like to invite you to revise the work to thoroughly address the reviewers' reports.As you will see below, the reviewers are generally positive about the manuscript and note that the study is well conducted. However, Reviewer #1 raises concerns with the modelling and quality of the density maps near the extracellular side of the receptor and Reviewer #2 notes that the rationale for the study should be more clearly framed in the Introduction and Discussion sections of the manuscript. Given the extent of revision needed, we cannot make a decision about publication until we have seen the revised manuscript and your response to the reviewers' comments to carefully document the map quality. Your revised manuscript is likely to be sent for further evaluation by all or a subset of the reviewers.plosbiology@plos.org) if you have any questions or concerns, or would like to request an extension. We expect to receive your revised manuscript within 3 months. Please email us  and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/*PLOS Data Policy*http://journals.plos.org/plosbiology/s/data-availability) requires that you make available all data used to draw the conclusions arrived at in your manuscript. If you have not already done so, you must include any data used in your manuscript either in appropriate repositories, within the body of the manuscript, or as supporting information . For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5Please note that as a condition of publication PLOS' data policy  such that it can be cited independently in the future. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at Thank you again for your submission to our journal. We hope that our editorial process has been constructive thus far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,RichardRichard Hodge, PhDSenior Editor, PLOS Biologyrhodge@plos.org------------------------------------REVIEWS:Reviewer #1: In the article "Structural basis of CMKLR1 signaling induced by chemerin9" Zhang et al. describes the human CMKLR1-Gi complex structure bound to chemerin9, MD simulations and functional assays together to investigate the activation mechanism. Upon receiving the manuscript, I was noted that a competing paper was already published in PNAS in March 2023. Below list some of my concerns over the model and interpretations.Major concern1. The overall model of CMKLR1 complex is well fit to density map, but near extracellular side there is a large amount of residues missing their density. It is particularly worrying that backbone density is also missing in some areas. This could significantly mislead readers or end users of the model. Some side chains should be trimmed to avoid confusions.2. It has been reiterated in the manuscript that the pi-pi interaction contributes to the binding of chemerin9 to the receptor. It appears to me that the authors seem to be abusing this term. For example, they stated "This residue and Phe150 of chemerin9 form π-π interactions with F2947.31  and F190ECL2 of CMKLR1, respectively". However, Phe150 is at least 12 Å away from F294. How is any interaction possible? In addition, there is merely no density to support the side chain of Phe150. If it was indeed a good pi-pi interaction between Phe150 and F190, why was it too difficult to see its density? See the screenshot below.Another example, "At the extracellular region of CMKLR1, the first N-terminal residue of chemerin9, Tyr149, forms a π-πinteraction with F2947.31 of CMKLR1". Although there might be a pi-pi interaction between two stated residues, it should be noted two residues at the so-called offset position are 6 Å apart. The authors need to double-check if such pi-pi interaction can be acclaimed. See the screenshot below.Also, "Phe156 of chemerin9 plays a crucial role in the interactions with the receptor, forming hydrophobic and π-π interactions with CMKLR1 residues F882.53, H952.60, M1233.36, Y2766.51, and I3067.43…". Please check if the orientation of Phe156 relative to F88 and Y276 aligns with the definition of pi-pi interaction. 3. The use of AlphaFold structure to represent the inactive state of CMKLR1 is unsound. First, the AlphaFold structure itself does not acclaim to be at the inactive state. Second, based on homology modeling, the source of initial models is unknown to users. Given these considerations, it is usually not used as an evidence to make comparisons between the active and inactive GPCR structures. Especially, discussing residue movements in so much details is not necessary. Minor concerns1. When discussing the simulation results, the authors seem to be quite surprised that chemerin9 as a potent agonist is able to stabilize the active state of CMKLR1 in the absence of Gi. The reasons for this uncommon view are neither explained in the manuscript nor found in the reference. 2. The authors also stated "This observation suggests that the active CMKLR1 conformation favors a close contact between the peptide agonist and the transmission switch motif." However, the two residues , Phe156 and W275, are at least 5.5 Å apart even when Gi is bound, which is not a close distance. Some could even say they do not contact one another. 3. The FSC cutoff for model-to-map is 0.5 instead of 0.143.4. Re-examining thoroughly how well the model is fit to density is necessary for this study. For example, Glu283 of the receptor is obviously laying outside its density.5. Comparisons between current study and previously published results in PNAS should be addressed in more volumes and figures. Reviewer #2 : The manuscript Structural basis of CMKLR1 signaling induced by chemerin9 reports the CryoEM structure of CMKLR1 aka Chem23R in complex with one of its peptide agonists. ChemR23 is implicated in inflammation and peripheral pain. In addition to its peptide agonist, it also suggested to mediate the effects of RvE1, a pro-resolving lipid. The authors showed that inducing primary human macrophages with chemerin results in a non-canonical phenotype of the macrophages, an observation consistent with its both pro-inflammation and pro-resolving roles reported in the literature. The primary focus of the manuscript is structural analysis of the CMKLR1/Gi complex. The authors obtained a high-resolution structure, performed mutagenesis of the key residues interacting with the ligand to confirm and delaminate their role in the ligand binding or in some case also being important for activation. They also performed limited structure-activity analysis of the chemerin9 peptide, and further investigated the roles of charged interactions by MD.Overall, this is an excellent structural biology study, providing the first experimental structure of the important pharmacological target, and a very detailed description of the ligand-binding site. Congratulations to the authors as obtaining a structure of GPCR/G protein complex is always challenging. It will be very useful for structure-based drug design of this important pharmacological target.Addressing a few minor points would really improve the readability of the manuscript and explain its importance to a non-expert.1. The problem statement - what exactly the authors were aiming to achieve needs to be clearly formulated. There are several problems mentioned in the introduction 1) The apparent contradictory functional roles of CMKLR1 … (2) However, it is not clear whether RvE1 candirectly bind to and activate CMKLR1 to induce Gi signaling and (3) However, the lack of synthetic ligands of CMKLR1. Which one of the problems? The solution is the structure but what was the problem?gpcrdb.org as of today, although this may not have been the case when the project was initiated. Some of the argumentation may need to be revised to reflect this. Depending on the problem statement the authors would choose, this may provide an opportunity to discuss drug design in structural context.2. There are 67 ligands reported for this receptor in 3. A potential binding site for RvE1 suggested on Fig 3b. Again, depending on the problem statement this could be given a more prominent position in the manuscript.4. Discussion: it is very concise but reads more like an introduction. It really needs to discuss how the new results addressed the problem statement stated in the beginning of the manuscript. Once the problem statement is clarified, it would be easier to focus the discussion. The discussion would also benefit from a short 3-4 lines conclusion summarising the key fundings.5. Technical comment: The details of staining of the receptor in microscopy  is not described anywhere. One can guess it is likely to be an anti-FLAG fluorescent Ab staining but this needs to be included in the methods.Reviewer #3: The manuscript by Zhang et al describes the cryo-EM structure of CMKLR with chimerin9 and a Gi heterotrimeric G protein. This is second structure (and paper) describing this complex, nevertheless it is a very well performed study that adds to the understanding of how chimerin9 binds to and activates CMKLR. The foundation for this study is the cryo-EM map of the complex that was determined to 2.9Å resolution. This map has excellent density for all parts of the ligand and coupled with well-performed modelling creates a solid foundation for the extensive mutagenesis and ligand structure-function studies authors undertook. With the exception of some minor points listed below, I fully recommend this manuscript for a publication in PLOS Biology.Minor comments :1. Cholesterol modelling in the map is a bit too ambitious- this density is too noisy/low contoured/uncertain to even assign a particular lipid 2. Confusing labels on Fig. S6 as some panels have G proteins and some do not.3. Fig. 6b- A bit weird that the cryo-EM structure did not capture the captured the closest distance of W273 and chimerin9 - a discussion sentence on that in the text would be very helpful.4. Could the explanation for "the CMKLR1-Gi complex, the Gi heterotrimer remained stably bound to the receptor, regardless of the presence of chemerin9 ." be that the simulation is too short? Authors only run the 100ns simulation, while it is my understanding that to assess G protein binding at G proteins generally longer time frames are required (1uM). Perhaps and alternative explanation can also be added/considered. 6 Sep 2023AttachmentResponsesToReviewers_ChemR23_2023_2.pdfSubmitted filename: Click here for additional data file. 17 Oct 2023Dear Dr Zhang,Thank you for your patience while we considered your revised manuscript "Structural basis of CMKLR1 signaling induced by chemerin9" for publication as a Research Article at PLOS Biology. Please accept my apologies for the delay in getting back to you during this round of the peer review process. This revised version of your manuscript has been evaluated by the PLOS Biology editors, the Academic Editor and the original reviewers.Based on the reviews, I am pleased to say that we are likely to accept this manuscript for publication, provided you satisfactorily address the remaining points raised by the reviewers. Please also make sure to address the following data and other policy-related requests that I have provided below (A-G):(A) We would like to suggest the following modification to the title: “"Structural basis of G-protein coupled receptor CMKLR1 activation and signaling induced by a chemerin-derived agonist”http://journals.plos.org/plosbiology/s/data-availability. For more information, please also see this editorial: http://dx.doi.org/10.1371/journal.pbio.1001797(B) You may be aware of the PLOS Data Policy, which requires that all data be made available without restriction: Note that we do not require all raw data. Rather, we ask that all individual quantitative observations that underlie the data summarized in the figures and results of your paper be made available in one of the following forms:-Supplementary files . Please ensure that all data files are uploaded as 'Supporting Information' and are invariably referred to  using the following format verbatim: S1 Data, S2 Data, etc. Multiple panels of a single or even several figures can be included as multiple sheets in one excel file that is saved using exactly the following convention: S1_Data.xlsx (using an underscore).-Deposition in a publicly available repository. Please also provide the accession code or a reviewer link so that we may view your data before publication. 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No significant changes were observed in the physical condition of the players at the different moments of the season. Moreover, a significant association was observed only between RPEresTOT-L and Repeated Sprint Ability standard deviation of 3 m (RSAsdec3m) . The results suggest that the competitive season represented considerable neuromuscular involvement in these players.The objectives of this study were to describe differentiated perceived training and match load (dRPE-L) of wheelchair basketball (WB) players during the whole season, to analyze the evolution of players’ physical condition changes during a full season and to analyze the association between dRPE-L and changes in physical condition during a full season. Nineteen Spanish Second Division WB players participated in this study. For a full season , dRPE-L was assessed with the session-RPE method, separating respiratory (RPEres-L) and muscular (RPEmus-L) perceived load. The physical condition of the players was also assessed at four different times during the season . The results showed a significantly higher total and average accumulated muscular RPE load (RPEmusTOT-L and RPEmusAVG-L) than total and average respiratory load (RPEresTOT-L and RPEresAVG-L) ( It is p players . In WB,  players . The Int players . After t players .Like other team sports , WB is aAs well as quantifying match or training physiological load, multiple field tests have been used in order to determine WB players’ physical condition, such as, speed, acceleration, change of direction ability (CODA), strength and endurance . UsuallyIn this line of research, the main objectives of the study were: 1) to describe the differentiated perceived training and match load (dRPE-L) of WB players during the whole season; 2) to analyze the evolution of the players’ physical condition throughout the season and 3) to analyze the association between the dRPE-L and the change in the players physical condition during the season.Nineteen Spanish Second Division WB players  with at least 2 years’ training experience in WB participated in this study. The participants were classified according to the Classification Committee of the IWBF  and had a valid license from the Spanish Federation of Sports for People with Physical Disabilities. All the values of the players who participated in the team training sessions and matches during the whole season were recorded and included for statistical analysis. This study was approved by the institutional research ethics committee, and all participants provided written informed consent as outlined in the Declaration of Helsinki (2013).The data were collected in the WB season for 10 months (September to June) . Over thThe session-RPE method was used to calculate perceived training and match load as proposed by t-test (rMAT) test and in the second session 3-3-6, 505 and Illinois Agility tests. All tests were performed on the same indoor track and in the same time slot (between 17 and 20 p.m.), with the individual competition wheelchair. All participants were instructed to perform the tests at maximum intensity. The 48 h prior to the tests, players did not perform any demanding physical activity. Before each testing session, players performed a standardized warm-up consisting of 5 min of wheelchair propulsion at low intensity, dynamic stretching and two acceleration drills.In order to analyze the players’ physical condition, they performed the same test battery at four different times during the season (T1-T4). The physical tests were performed in two different sessions, separated by at least 48 h in the same week. In the first session, the players performed the Repeated Sprint Ability (RSA) and Repeated Modified Agility Three to six linear sprint test: The players had to perform a 12 m sprint divided into three short sprints of 3 m, 3 m and 6 m in a straight line . Partici505 test: The previously proposed protocol for WB players was used (was used . After pIllinois Agility Test (IAT): The players had to perform the protocol previously proposed for wheelchair athletes (athletes . ParticiRepeated Sprint Ability test (RSA): A protocol previously used in WB players was employed (employed . Particiemployed . RSASdecemployed : RSASdecRepeated Modified Agility T-test (rMAT): Players performed the protocol previously used by other authors in WB  in terms of RPEres-L and RPEmus-L, and the results of each of the tests at the four measuring times . A paired samples t-test was performed to determine the differences between RPEres-L and RPEmus-L in each period. In each case, the mean differences were calculated as a percentage (Δ%) = [(B–A) x 100/A]. Also, the effect size (ES) . The p < 0.05 criterion was used for establishing statistical significance.Standard statistical methods were used to calculate of the mean and standard deviation (SD). Data were screened for normality of distribution and homogeneity of variances using the Shapiro-Wilk normality test and Levene test. A repeated measures ANOVA with Bonferroni ize (ES)  was calcize (ES) . Data anp < 0.01; ∆% = 33.2; ES = 0.55, large). In this vein, players also reported a significantly higher perceived load in RPEmusAVG than RPEresAVG . The dRPE-L values of each week across the whole WB season are shown in p < 0.05, ∆% = 11.2–99.3; ES = 0.40–1.64, small–large).Team RPEresTOT-L and RPEmusTOT-L recorded during the whole season was 24,985.3 ± 11,526.1 AU and 33,265.3 ± 17,741.3 AU, respectively, while the RPEresAVG-L and dRPEmus-AVG-L was 1,050.5 ± 494.5 AU and 1,376.2 ± 741.9 AU. A significantly higher perceived load was observed in RPEmusTOT-L than RPEresTOT-L , at P2 than at P1  and at P2 than at P3 . In the weekly average, although no differences were observed between the different periods in RPEresAVG-L, RPEmusAVG-L was higher in P1 with respect to P2  and P3  and higher in P2 than in P3 . Significant differences were observed between RPEresTOT-L and RPEmusTOT-L, and between RPEresAVG-L and RPEmusAVG-L in P1  and P2 .Team total and weekly average dRPE-L for each period is presented in p < 0.05, ∆% = −7.49, ES = −0.80, very large).p < 0.05). At P1, a significant association was observed between total and mean RPEres-L with RSASdec1m, RSASdec3m and RSASdec5m . In P2, on the other hand, RPEres-L did not correlate with any variable while average and total RPEmus-L was related to RSAavg3m and RSAavg5m . Finally, in the latter part of the season (P3), RPEres-L and RPEmus-L did not correlate with any change in physical condition.A significant association between total and average dRPE-L and physical condition delta value was found only in RSA during all periods without a clear pattern. A significant association was observed between the RPEresTOT-L of the whole season with the change in RSAbest5m between T1 vs T4  of WB players during the whole season, to analyze the evolution of players’ physical condition changes during a full season and to analyze the association between dRPE-L and changes in physical condition during the season. Considering the scant evidence of longitudinal studies analyzing the effects of internal load on physical condition in WB, the main objectives of this study were to describe dRPE-L and physical condition changes during a WB full season and to analyze the association between dRPE-L and changes in physical condition during the season. The main findings were: a) a significantly higher dRPE-L was observed in P1 than P2 and P3, as well as in P2 than in P3 and the muscular perceived load being generally significantly higher than the respiratory perceived load in all periods; b) there were no significant changes in relation to physical condition except for the RSASdec3m; and c) significant associations were observed between RPEmus-L with RSAavg3m and RSAavg5m. To the best of the authors’ knowledge, this is the first study reporting relationships among the perceived load and physical condition changes in WB players for a whole competition season.The dRPE-L is a practical tool to assess the internal load of training and matches . RegardiThe 3-3-6 linear sprint Test , 505 TesQuantifying and modelling the training load in team sports such as WB is an important aspect in order to know the appropriate training stimulus for each player and to avoid the negative consequences of an excessive training load  . While sSome limitations of this study must be taken into consideration. The results of this study should be taken with caution when extrapolating them to another team, as each player, depending on gender, type of disability or performance level, may behave differently. In this sense, the level of the team opponent could affect the internal load recorded during the matches. Therefore, it could be interesting to analyze this issue in future research. Although the RPE is a useful tool for quantifying training and match load, it is affected by personality, player characteristics, environmental and cognitive factors . ConsideThe results of this study show significant changes in the perceived load (RPEmus-L) between the different periods throughout the season. In terms of physical condition, significant differences were only observed in some RSA variables at different times during the season. Despite the fluctuation in perceived load, significant associations were observed anecdotally between perceived load and some RSA variables. In future studies it could be interesting to analyze this association considering the characteristics of the WB players .


Schizophrenia is a severe and persistent mental condition that causes disability. For subsequent clinical care, it is extremely practical to effectively differentiate between patients who respond to therapy quickly and those who do not. This study set out to document the prevalence and risk factors for patient early non-response.The current study included 143 individuals with first-treatment and drug-naïve (FTDN) schizophrenia. Patients were classified as early non-responders based on a Positive and Negative Symptom Scale (PANSS) score reduction of less than 20% after 2 weeks of treatment, otherwise as early responders. Clinical subgroups’ differences in demographic data and general clinical data were compared, and variables related to early non-response to therapy were examined.Two weeks later, a total of 73 patients were described as early non-responders, with an incidence of 51.05%. The early non-response subgroup had significantly higher PANSS scores, Positive symptom subscale (PSS) scores, General psychopathology subscale (GPS) scores, Clinical global impression scale - severity of illness (CGI-SI) and Fasting blood glucose (FBG) levels compared to the early-response subgroup. CGI-SI and FBG were risk factors for early non-response.High rates of early non-response have been seen in FTDN schizophrenia patients, and risk variables for predicting early non-response include CGI-SI scores and FBG levels. However, we need more in-depth studies to confirm the generalizable range of these two parameters. The medEarly response to treatment in schizophrenia is of great significance for better management of the disorder in the future and is touted as one of the most reliable predictors of long-term clinical outcome in schizophrenia . Poor eaAlthough the incidence of early unresponsive patients with schizophrenia has been reported, the known reported results vary considerably due to differences in inclusion criteria and study objectives, and incidence rates from China are lacking. For example, German researchers reported an incidence of 43.43% , compare2.2.1.For the present study, 143 patients with first-treatment and drug-naïve (FTDN) schizophrenia admitted to Wuhan Mental Health Centre from May 2019 to September 2020 were selected.Patients must meet the following inclusion criteria:1. Meet the diagnostic criteria for schizophrenia according to the International Classification of Diseases, 10th Revision (ICD-10), and the total duration of the illness should not exceed 6 years.2. No use of antipsychotic medication prior to the first hospitalization, or the duration of medication did not exceed 1 week.3. The included samples were all Chinese Han.4. Male or female, aged 18–45 years.5. Positive and Negative Symptom Scale (PANSS) total score was more than 60 points.Exclusion criteria: excluding breastfeeding and pregnant women, severe somatic diseases confirmed as diabetes, hypertension and diseases of the immune system; excluding bipolar disorder, intellectual disability, mental disorder due to epilepsy, major depressive disorder and other mental diseases.The study was reviewed and approved by the Ethics Committee of Wuhan Mental Health Center, and all participants signed a written informed consent form.2.2.The study, which was intended to be a real-world study, calculated the incidence of early responders to treatment in schizophrenia patients who met the inclusion criteria within a specific time frame, compared the variations in demographic data and general clinical parameters between the two clinical subgroups of early-response and early non-response, and examined the factors associated with the early-response group. Finally, we plot receiver operating characteristic (ROC) curves for the acquired correlates and determine the cut-off values that can be used to differentiate treatment response.We have designed our own EXCLE spreadsheet for recording demographic and general clinical information on patients admitted to the hospital . We collMedication: Medication: The investigators did not interfere with the clinically prescribed medication for the patients, and the bedside doctors flexibly selected the type of antipsychotic medication for the included patients based on the patient’s particular circumstances and their individual clinical experience. The bedside physician may titrate the chosen antipsychotic drug for 2 weeks while adhering to the prescribed guidelines and taking into account the patient’s unique circumstances, but without changing the medicine.nd weekend score) / (baseline score - 30) × 100%. The PANSS scores were also administered to the included patients by 2 professionally trained attending psychiatrists on 2 successive occasions (day of patient admission and 2 weeks after treatment). Also, the included patients received the PANSS ratings from 2 attending psychiatrists with professional training on 2 separate dates (day of patient admission and 2 weeks after treatment).Clinical subgroup classification rules: We distinguished between two clinical subgroups using the PANSS subtraction rate. Patients were categorized as part of the early-response group after 2 weeks of therapy if their PANSS decrease rate was more than or equal to 20%; otherwise, they were part of the early non-response group . The PAN2.3.p < 0.05 (two tails).Data obtained for continuous measurements with normal distribution are expressed as means and standard deviations, and categorical variables are expressed as counts. Independent samples t-test is used to compare data from different groups. The chi-square test or Fisher’s exact probability was used to compare rates. We then constructed a binary logistic regression model with early non-response as the outcome variable and parameters that differed in the univariate analysis as independent variables to identify factors associated with the early nonresponse group. Finally, we plotted ROC curves for the factors obtained from the logistic regression that influenced early non-response, which were used to determine meaningful cut-off values. IBM SPSS  was used for data analysis. GraphPad Prism software  was used for plotting. The significance level for all statistical tests was set at 3.3.1.p = 0.020), PSS scores (p = 0.007), GPS scores (p = 0.020), CGI-SI scores (p = 0.003), and FBG (p = 0.003).The average doses of the seven antipsychotic drugs at the end of 2 weeks were aripiprazole (23.75 ± 4.95) mg, olanzapine (17.78 ± 3.49) mg, quetiapine (706.82 ± 67.78), risperidone (5.26 ± 0.90) mg, ziprasidone (129.09 ± 22.02) mg, perphenazine (25.33 ± 7.21) mg and haloperidol (25.83 ± 3.66) mg. 2 weeks later, a total of 70 included patients were classified in the early-response group, with a reduction rate of (37.07 ± 14.05) %, while another 73 patients were classified in the early non-response group, with a mean reduction rate of (8.77 ± 6.44) %. The percentage of the early non-response group was 51.05%. Compared to the early-response group, the early non-response group had significantly higher PANSS scores , and FBG  were risk factors for early non-response.Next, we focused on the related factors of early-response group. A binary logistic regression model  was constructed with early non-responder as the outcome variable and parameters with differences in univariate analysis as independent variables. As shown in 3.3.p = 0.005, 95% CI = 0.544–0.726) and FBG was 0.637 . According to the Youden index, the optimal cut-off value for CGI-SI was 6  and the optimal cut-off value for FBG was 4.53 mmol/l .Finally, we plotted the ROC curves for both CGI-SI and FBG variables, as shown in 4.We provide genuine information on the clinical incidence and variables linked to early non-response in FTDN schizophrenia patients as part of a real-world clinical investigation. The following are the study’s primary conclusions: 1. The PANSS scores (both PSS and GPS scores), CGI-SI scores, and fasting blood glucose levels were all higher in the clinical subgroup of early non-response with FTDN schizophrenia than they were in the clinical subgroup of early-response. 2. The early non-response was predicted by the CGI-SI scores and FBG levels. 3.The optimal cut-off values for the two parameters of 6 for CGI-SI and 4.53 mmol/l for FBG.We discovered that the severity of psychopathology was higher in FTDN schizophrenia patients categorized as early non-responders after 2 weeks of treatment. Two clinical studies, which strictly limited prescribed medications, reported differing results from ours, finding that the early non-response subgroup had significantly higher disease severity at baseline  in first-episode schizophrenia patients , 16. Howa priori, and we reported a 51.05% incidence of early non-response, more than half of the total sample, and reported differently in all the above studies.The available known studies that focus on the incidence of early non-response to treatment for schizophrenia report a high degree of heterogeneity, and almost without exception these have been conducted under the premise of setting strict antipsychotic drug classes as inclusion criteria, with few real-world studies involved. A clinical trial from South Africa prescribing long-acting flupenthixol injections to patients with first-episode schizophrenia reported a 12% (15/126) incidence of treatment non-response . AnotherIn addition to the impact of differences in study design and enrolment criteria on response rates, the diversity of instruments and assessment methods used to assess response rates is one of the key influences on the heterogeneity of response rates. For example, a large German study reported early non-response rates in 528 severely impaired schizophrenic patients using four different assessment tools, respectively, an incidence of 30% reported using the PANSS positive score, 41% reported using the PANSS negative score, and the CGI-severity score reported at 63% and Scale of Occupational and Functional Assessment (SOFAS) reported at 35% . In paraAs with incidence, there are similarly no more consistent and universally accepted conclusions about the predictors of early non-response to treatment for schizophrenia. There are many factors that influence the prognosis and outcome of people with first-episode schizophrenia, such as duration of untreated after the episode , 27, degc-statistics = 0.85, 95% CI 0.77, 0.93; boostrap estimate 0.8598) is a predictor of good response to treatment at 6 months  as the target variable, found that baseline GAF score .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Lactiplantibacillus plantarum Q7 and L. plantarum F3-2 on the intestinal barrier of mice were explored. It was shown that L. plantarum Q7 promoted the expression of mucin MUC2 to enhance the protection provided by the intestinal mucus layer. L. plantarum Q7 up-regulated the gene expression of intestinal tight junction proteins ZO-1 and JAM-1 significantly, and L. plantarum F3-2 up-regulated ZO-1 and Claudin-1 markedly, which exhibited tight junction intestinal barrier function. The two strains promoted the release of IgA and IgG at varying degrees. The antimicrobial peptide gene RegIIIγ was up-regulated markedly, and the gene expression of inflammatory cytokines appeared to exhibit an upward trend with L. plantarum Q7 treatment, so as to enhance intestinal immune regulation function. Furthermore, L. plantarum Q7 and L. plantarum F3-2 increased the abundance of the beneficial bacteria Muribaculaceae, inhibited the growth of the harmful bacteria Parabacteroides, and facilitated the synthesis of total short-chain fatty acids (SCFAs), which seemed to favor the prevention of metabolic diseases. Our results suggested that L. plantarum Q7 and L. plantarum F3-2 showed strain specificity in their protective effects on the intestinal chemical, physical, immunological and biological barriers of mice, which provided theoretical support for the selective utilization of bacteriocin-producing strains to regulate host health.Bacteriocins are crucial metabolites of probiotics that display beneficial functions. The intestinal barrier is an important target on which probiotics exert their intestinal health activity. However, the impacts of bacteriocin-producing probiotics on the intestinal barrier are unclear. In this study, the effects of bacteriocin-producing  Probiotics have been confirmed as active microorganisms that play advantageous roles in the body when adequate amounts are ingested . In receL. plantarum GL17 and Lactiplantibacillus brevis AY858 enhanced the expression of tight junction proteins such as ZO-1, Occluding and Claudin-1 to improve the integrity of the gut barrier and protect mice from intestinal inflammation. The intestinal immunological barrier is essential for appropriate immune defense and inflammation control. Probiotics can stimulate the anti-inflammatory or pro-inflammatory immune response in the intestine to enhance immunity function [L. plantarum HAC01 administration regulated the intestinal microbiota, thereby ameliorating metabolic syndrome in mice [The intestinal chemical barrier is mainly formed of the mucus layer. When the intestinal epithelium is invaded by pathogenic microorganisms, some probiotics can induce epithelial cells to secrete mucins that reduce the binding of intestinal pathogens to mucosal epithelial cells, which strengthens the intestinal mucosal barrier’s function . As an ifunction , and thefunction . Intestifunction . It was  in mice .Bacteriocins are important metabolites produced by probiotics and exhibit biological antibacterial, anti-infection, anti-inflammation and immunomodulation activities . The maiL. plantarum Q7 and L. plantarum F3-2 have been proven to exhibit a bacteriocin-producing capacity and probiotic potential in vitro [L. plantarum Q7 and L. plantarum F3-2 on the intestinal chemical, physical, immunological and biological barriers in vivo, which would provide a scientific basis for the development of safe and effective bacteriocin-producing probiotics.Our preliminary research showed that in vitro . In thisL. plantarum Q7 and L. plantarum F3-2 were isolated from yak milk and infant feces, respectively. The two strains were cultured in de Man, Rogosa, and Sharpe (MRS) broth  at a 2% (v/v) inoculation amount and activated twice at 37 °C for 24 h. The bacterial cells were collected via centrifugation  and washed twice with phosphate-buffered saline (PBS), after which deposits were dissolved in PBS, and the concentration was 1 × 109 CFU/mL.Specific pathogen-free (SPF) C57BL/6J mice  were used in the experiment. All mice were kept in an animal care facility  and sufficient food and water were provided. Mice were acclimated to the environment for 7 days, and then randomly assigned to 3 groups (n = 12 in each group): the control group (control), which was treated with 200 μL PBS, and the L. plantarum Q7 group (Q7) and L. plantarum F3-2 group (F3-2), which were treated with 200 μL L. plantarum Q7 or L. plantarum F3-2 suspension, respectively. The total oral administration duration was 4 weeks. Mice were weighed weekly, and food and water consumption were recorded every 3 days. Fecal samples were collected from each mouse on the last day of oral gavage. After mice were anesthetized and euthanized, eyeball blood was extracted, which was allowed to stand for 2 h, and then centrifuged  to obtain serum. All experiments were approved and supervised by Animal Ethics Committee of Ocean University of China (permission number: SPXY2022030802).After the ileal and colonic tissues were fixed in 4% paraformaldehyde  and dehydrated, the paraffin-embedded sections were dewaxed, stained with hematoxylin and eosin (H&E) , and visualized using a microscope . For the immunohistochemical evaluation of mucin MUC2 expression, the colonic sections were subjected to antigen repair, and 3% hydrogen peroxide solution  was added to block endogenous peroxidase activity. The tissues were sealed with 3% BSA  at room temperature for 30 min, and then incubated with a primary antibody overnight at 4 °C. After incubation with a secondary antibody, the sections were stained with DAB  and hematoxylin, and observed under a microscope.The ileum and colon were homogenized in RIPA lysis buffer  using a tissue grinder  prior to centrifugation . The supernatants were obtained and the levels of IgA and IgG in the serum, ileum and colon were measured following the directions of the ELISA kit .−ΔΔCt method was utilized to calculate the relative expression of genes. The specific primers for RT-qPCR are listed in Total RNA was extracted from the ileal and colonic tissues with TRNzol Universal , and cDNA was synthesized using ReverTra Ace qPCR RT Master Mix with gDNA Remover . Genes were quantitated on a CFX96 Real-Time PCR System  in a volume of 25 μL. β-actin was used as a housekeeping gene and the 2The extracted genomic DNA samples from mice feces were used as the template to amplify the V3-V4 regions of bacterial 16S rRNA genes using the forward primer 338F (ACTCCTACGGGAGGCAGCA) and reverse primer 806R (GGACTACHVGGGTWTCTAAT). The libraries were built using TruSeq Nano DNA LT Library Prep Kit , and qualified libraries were underwent paired-end 2 × 250 bp sequencing on an Illumina NovaSeq platform. The high-quality sequences were assigned to OTUs with 97% clustering thresholds by UCLUST. The sequence data were analyzed using QIIME2 2019.4 software and the R package.g, 10 min, 4 °C). The supernatants were taken and vortexed with anhydrous ether  at 1: 1 (v/v), after which the samples were centrifuged , and the ether layer was collected for analysis. SCFAs were separated using an HP-FFAP column . The initial temperature was 90 °C, which was elevated to 150 °C at 12 °C/min. Then, the temperature was elevated to 220 °C at 20 °C/min and held for 4.5 min. Acetic acid, propionic acid and butyric acid  were the standard solutions.SCFAs in fecal samples were determined via gas chromatography–mass spectrometry  using the method of Han et al.  with somt-test was used for comparing statistical differences between two groups, and multiple comparisons were evaluated by ANOVA and Duncan’s test using IBM SPSS Statistics 22.0. The criterion for significance was p < 0.05.All values were expressed as mean ± SD. An independent-samples L. plantarum Q7 and L. plantarum F3-2 were orally administered to mice, the changes in the body weight, food and water intake of mice were monitored, and the results were shown in p > 0.05). The intragastric administration of the two strains did not lead to any changes in the food and water consumption of mice (p > 0.05).The procedure of the animal experiment was indicated in L. plantarum on the intestinal chemical barrier, intestinal mucin MUC2 expression in mice was measured. It was illustrated in Muc2 in the ileum and colon were up-regulated remarkably with the oral administration of L. plantarum Q7 (p < 0.05), while L. plantarum F3-2 intervention had no evident effect on Muc2 (p > 0.05). Further analysis was conducted via immunohistochemistry. After the mice were gavaged with L. plantarum Q7, the positive area of MUC2 in colon was increased significantly (p < 0.05), while there was no significant difference between the F3-2 group and the control group (p > 0.05), which was in line with the result of Muc2 gene expression , and the crypt depth remained almost unchanged (p > 0.05) when mice were fed with L. plantarum Q7 and L. plantarum F3-2 (p > 0.05). The gene expression levels of the tight junction proteins ZO-1, JAM-1 and Claudin-1 in the ileum and colon were measured. L. plantarum Q7 treatment significantly up-regulated the gene expression of ZO-1 and JAM-1 (p < 0.05), and L. plantarum F3-2 treatment significantly up-regulated ZO-1 and Claudin-1 (p < 0.05).The effects of two strains of ol group A. The virum F3-2 . There wL. plantarum strains in the intestinal immunological barrier. It can be seen from p < 0.05). The contents of IgA in the ileum (p < 0.01) and colon (p < 0.05) were remarkably up-regulated in the Q7 group, but no obvious change occurred in the IgA concentration in mice intestine in the F3-2 group (p > 0.05). Moreover, the IgG contents in the serum (p < 0.01), ileum (p < 0.01) and colon (p < 0.05) were significantly increased when L. plantarum Q7 was administered, while L. plantarum F3-2 intervention only remarkably increased IgG content in the colon of mice (p < 0.05). The gene expression of RegIIIγ was shown in RegIIIγ gene expression in the ileum and colon of mice gavaged with L. plantarum Q7 (p < 0.05), but L. plantarum F3-2 had no significant influence on RegIIIγ (p > 0.05). Furthermore, the gene expression levels of six inflammatory cytokines in the ileum and colon were detected. TNF-α, IL-6, IFN-γ, IL-10, IL-12 and IL-1β among the three groups (p > 0.05). However, it was worth mentioning that the gene expression of the above inflammatory cytokines showed an upward trend in the Q7 group compared with that of the control group.The determination of immunoglobulins, endogenous antibacterial peptide and inflammatory cytokines in the intestine of mice was carried out to assess the roles of the two L. plantarum strains on the intestinal biological barrier. As shown in p > 0.05)  in the feces of mice showed a decreasing trend (p > 0.05) , and Muribaculaceae abundance showed an upward trend with L. plantarum F3-2 intervention (p > 0.05) , propionic acid (p < 0.05) and total SCFAs (p < 0.01) in the Q7 group were significantly higher than those in the control group. After mice received L. plantarum F3-2 via oral gavage, the concentration of acetic acid showed a rising trend (p > 0.05), and the content of total SCFAs was increased significantly compared to the control group (p < 0.05).The fecal microbiota of mice was analyzed to investigate the effects of the two  > 0.05) B. PCoA w > 0.05) C, indica > 0.05) E. In thetarum Q7 F. The di > 0.05) H. Furthetarum Q7 I. In conectively J. Parasuentation K. The efPediococcus acidilactici UL5 to mice reduced the viable counts of the pathogen Listeria monocytogenes in the intestine, which would basically not perturb the gut microbial equilibrium. The bacteriocin-producing L. plantarum NCIMB8826 has been shown to reduce the levels of pro-inflammatory cytokines TNF-α and IL-6 in IBD mice [L. plantarum Q7 and L. plantarum F3-2 with probiotic potential were given to mice via intragastric administration, and it was found that the two strains did not affect the body weight and basic physiological activity of mice, such as feeding and drinking, so they were preliminarily regarded as safe organisms, and their effects on the intestinal barrier of mice were further investigated.A large number of bacteriocins from lactic acid bacteria have been identified, and they are mainly used as biological preservatives in food preservation studies and to alleviate pathogens infection in host . With thIBD mice . In viewL. plantarum Q7 promoted the expression of mucin MUC2, which was conductive to lubricating the intestinal tract, providing adhesion sites for the gut symbiotic microbiota, and resisting pathogens invasion, thereby preventing inflammation and damage, and protecting the intestinal chemical barrier. However, L. plantarum F3-2 had no significant influence on MUC2, which revealed differences in the effects of various bacteriocin-producing strains on the intestinal chemical barrier. In terms of the intestinal physical barrier, the two strains were helpful in maintaining good colonic tissue morphology in mice, and improving the ratio of villus height to crypt depth of the ileum, which enhanced the intestinal absorption of nutrients. Tight junction proteins are the most vital structures of the intestinal physical barrier. The gene expression of ZO-1 and JAM-1 in the intestine of mice were remarkably up-regulated with L. plantarum Q7 intervention, and ZO-1 and Claudin-1 showed a significant increase after L. plantarum F3-2 supplementation, indicating that bacteriocin-producing L. plantarum could protect the intestinal physical barrier by promoting tight junction proteins expression, which was in accordance with the study of Heeney et al. [The intestinal chemical barrier is mainly composed of the mucus layer. MUC2 is the main component of the gut mucus layer, and covers the top of the intestinal epithelium to play a crucial role in enhancing mucosal immunoregulatory and maintaining intestinal homeostasis . L. plany et al. .L. plantarum on the intestinal immunological barrier were explored, the results revealed that the two strains promoted the release of IgA and IgG in the serum and intestine to varying degrees, which suggested that bacteriocin-producing strains could strengthen the intestinal immune function, but there existed differences when different strains acted on immunoglobulins in distinct tissues of mice. Antimicrobial peptides are crucial participants in host natural defense and play a significant part in innate immunity [L. plantarum Q7 remarkably up-regulated the gene expression of RegIIIγ in the ileum and colon of mice to enhance the antibacterial, anti-inflammatory and immunomodulatory functions of the intestine, which demonstrated that the intervention of exogenous bacteriocin-producing strains could affect the immune function of the host by regulating the expression of endogenous antimicrobial peptides. In addition to immunoglobulins and antimicrobial peptides, inflammatory cytokines are also closely related to immune function. With the increasing studies on bacteriocins regulating cytokines secretion, bacteriocins have been confirmed to promote the production of inflammatory cytokines to enhance body immunity, and decrease proinflammatory cytokine levels to develop anti-inflammatory function [+ CD8+ T cells proportion, but when PBMC was stimulated by lipopolysaccharide (LPS), Nisin reduced the production of LPS-induced proinflammatory factor IL-6, reflecting the dual roles of Nisin in immunomodulation and anti-inflammation. In our study, the two strains had little effect on intestinal inflammatory cytokines in mice, but the gene expression of inflammatory cytokines showed an upward trend after the oral administration of L. plantarum Q7. Cui et al. [Pediococcus pentosaceus treatment in the short term promoted an increase in inflammatory cytokines in the colon of healthy mice, which initiated the immune response and activated the immune regulation system. Similarly, the up-regulation trend of L. plantarum Q7 on inflammatory cytokines reflected its potential to enhance body immunity and treat immunodeficiency. However, it is necessary to further analyze the anti-inflammatory activity of strains in a state of inflammation, so as to evaluate the immunomodulatory function of strains comprehensively. The two bacteriocin-producing strains indicated strain specificity in terms of their influences on intestinal immunity. In particular, L. plantarum Q7 increased the contents of immunoglobulins and the expression level of antimicrobial peptide without causing an inflammatory response, which was a critical way to protect the intestinal immunological barrier in mice.When the effects of bacteriocin-producing immunity . The antimmunity . L. planfunction . Małaczefunction  found thi et al.  reportedP. acidilactici with bacteriocin production ability could significantly enhance the Chao 1 index of the intestinal microbiota in mice, but the Shannon index showed an insignificant downward trend. Hence, it can be concluded that different bacteriocin-producing lactic acid bacteria had various effects on the α-diversity of intestinal communities.The intestinal biological barrier mainly consists of gut microorganisms. The oral delivery of two strains led to a slight increase in Chao 1, Faith_pd, Shannon and Observed_species, suggesting that these strains slightly increased the richness, uniformity and diversity of the gut microbiota with no significant difference, which was slightly differentiated from previous research. Qiao et al.  found thL. plantarum Q7 and L. plantarum F3-2 had certain impacts on the composition and structure of the intestinal microbiota. After the intervention of the two strains, F/B in the intestine of mice tended to be decreased. It was reported that the intake of Lactobacillus could help reduce F/B, thereby inhibiting the occurrence of metabolic diseases [Proteobacteria is regarded as a signature of dysbiosis and disease risk [L. plantarum Q7 reduced Proteobacteria abundance to inhibit intestinal pathogens growth, which might be attributed to the production of bacteriocin from L. plantarum Q7. Muribaculaceae and Akkermansia are beneficial bacteria in mice feces, and were up-regulated in the Q7 group. The regulation of the abundance of Muribaculaceae and Akkermansia could provide a basis for ameliorating inflammation and metabolic diseases such as obesity [Lactiplantibacillus acidophilus JCM 1132 significantly increased Akkermansia abundance in the intestinal tract of mice after a one-week withdrawal period, and it was speculated that bacteriocin might have induced the change of Akkermansia by killing specific bacteria [Akkermansia can degrade mucin specifically to stimulate the secretion of new mucin and promote MUC2 expression, which is essential for accelerating intestinal epithelial regeneration and repairing damaged intestinal mucosa [Muc2 was markedly up-regulated in the Q7 group, which might be associated with the increasing abundance of Akkermansia in the intestinal microbiota due to L. plantarum Q7 treatment. Parabacteroides and Parasutterella are potentially harmful bacteria. An increase in Parabacteroides abundance can exacerbate infection and increase the permeability of the intestine [Parasutterella will bring about a decrease in gut microbiota diversity or microecological dysbiosis, which is associated with the occurrence of irritable bowel syndrome, obesity and type II diabetes [L. plantarum strains were conductive to improving the structure of the intestinal microbiota in healthy mice, but there also existed certain differences in the regulation effects of different strains, which was also proposed by Qiao et al. [Lactiplantibacillus salivarius UCC118 had a subtle influence on the mice microbiota via a partial bacteriocin-dependent mechanism. Gebhart et al. [Clostridium difficile from colonizing in the host intestine without disrupting protective indigenous microbiota. The above studies showed that bacteriocins and bacteriocin-producing probiotics promoted subtle but favorable changes in intestinal communities structure without destroying the original protective microbiota, which was in line with our study.According to the results of β-diversity, diseases , which iase risk . L. plan obesity . Similarbacteria . Moreovel mucosa . In our ntestine . An incrdiabetes . The twoo et al.  previouso et al.  pointed t et al.  obtainedMuribaculaceae abundance was correlated with propionic acid. It was demonstrated that Parasutterella abundance was markedly lower based on the accumulation of total SCFAs in the feces of beagle dogs [L. plantarum Q7 and L. plantarum F3-2 regulated the gut microbiota to affect the production of SCFAs, thus protecting the intestinal biological barrier.As the main energy source of the intestinal epithelium, SCFAs play a critical role in protecting the intestinal barrier. The two strains increased the contents of SCFAs, which contributed to regulating host immunity, inflammatory response and energy metabolism. Smith et al.  reportedgle dogs . TherefoL. plantarum Q7 and L. plantarum F3-2 could protect the intestinal chemical, physical, immunological and biological barriers of healthy mice at different degrees. The effects of L. plantarum Q7 were better than those of L. plantarum F3-2 regarding the levels of mucin, immunoglobulins, antibacterial peptide and SCFAs. To fully evaluate the impacts and mechanisms of bacteriocin-producing probiotics on the intestinal barrier, further research is required to explore the injury repair effects of L. plantarum Q7 and its mutant strain with knock-out of the gene related to bacteriocin production on the intestinal barrier of mice with intestinal dysfunction. These findings would provide a reference for the development of different bacteriocin-producing probiotics with beneficial characteristics to strengthen intestinal barrier function.In conclusion, our study demonstrated that


No significant differences were observed in ventilation or CPR quality. In conclusion, lifeguards in good physical condition can perform simulated infant CPR of a similar quality to that of CPR carried out on a victim who is lying down in a fixed position.The aim of this study was to compare the quality of standard infant CPR with CPR in motion  via performing maneuvers and evacuating the infant from a beach. Thirteen trained lifeguards participated in a randomized crossover study. Each rescuer individually performed three tests of 2 min each. Five rescue breaths and cycles of 30 chest compressions followed by two breaths were performed. Mouth-to-mouth-and-nose ventilation was carried out, and chest compressions were performed using the two-fingers technique. The manikin was carried on the rescuer’s forearm with the head in the distal position. The analysis variables included compression, ventilation, and CPR quality variables, as well as physiological and effort parameters. Significantly lower compression quality values were obtained in running CPR versus standard CPR (53% ± 14% versus 63% ± 15%;  In the event of a pediatric cardiac arrest taking place in a dangerous situation or at a remote location, it is essential to evacuate the victim as soon as possible. However, it is still critical to begin resuscitation maneuvers early in order to improve the chances of survival ,2,3,4. TSince the 1990s, the American Heart Association (AHA) has advised that it is possible to carry a pediatric victim (infant) in arms while the steps of cardiopulmonary resuscitation (CPR) are initiated . A studyTo minimize response time, a pediatric victim could be carried in the rescuer’s arms; then, resuscitation maneuvers would be performed while running and evacuating the victim. However, we do not know if the time gained via running would be worthwhile if the CPR quality deteriorated greatly. To our knowledge, no study published to date has researched this topic. We hypothesized that lifeguards in good physical condition who were trained in basic life support would be able to perform quality CPR. Therefore, this research was carried out with the aim of comparing the quality of standard CPR (S-CPR) versus walking CPR (W-CPR) and running CPR (R-CPR) in a simulated scenario on a beach.A randomized crossover simulation study was used to compare standard CPR (S-CPR) with an infant manikin on a firm surface versus walking CPR (W-CPR) and running CPR (R-CPR) with an infant manikin on the rescuer’s forearm while performing the maneuvers of resuscitation (chest compressions and mouth-to-mouth-and-nose ventilation) .Thirteen lifeguards were asked to perform 2 min of CPR on an infant manikin in three different scenarios. The young and healthy rescuers were in a good physical condition suitable for water rescue; lifeguards undergo physical tests every year for the renewal of their contract, and all participants had a valid professional license at the time of the research. In addition, the lifeguards made a subjective assessment of their physical condition on a 0–10 scale.Before the investigation, all participants had attended a one-hour training course in basic pediatric life support. Participants trained individually, using standard CPR on an infant simulator while obtaining feedback, until they were able to perform quality CPR over 70%) [0% [7]. TAll participants were informed about the study and gave their written informed consent. The research respected the ethical principles of the Declaration of Helsinki.Each participant randomly performed 2 min of CPR in three situations: (A) S-CPR, (B) W-CPR, and (C) R-CPR. During the S-CPR test the manikin was on a firm surface. In the W-CPR and R-CPR tests the infant manikin was carried on the rescuer’s forearm with the head in the distal position. Rescuers did not receive any feedback during the tests.(1)S-CPR/W-CPR/R-CPR(2)S-CPR/R-CPR/W-CPR(3)W-CPR/S-CPR/R-CPR(4)W-CPR/R-CPR/S-CPR(5)R-CPR/S-CPR/W-CPR(6)R-CPR/W-CPR/S-CPRThe order of the three tests performed by each participant was randomized to minimize possible biases related to previous experience. A randomization by blocks was performed, so that the following order blocks were created:www.randomizer.org, accessed on 21 February 2020) computer software was used to assign the order in which the tests would be performed.After this block selection, each participant was randomly assigned to one of the blocks. Research Randomizer (The recommendations of the European Resuscitation Council (ERC) 2021  for infaA minimum rest period of 1 h was set between each test . After eData capture was carried out on Mogor Beach in Marín (Northwest Spain), beginning at the following GPS location: 42°23′09.4″ N–8°43′09.8″ W.Two groups of variables were evaluated.Chest Compressions (CC): Number of CC; Mean depth in mm; Mean rate in CC/min; Percentage of CC with adequate depth; Percentage of CC with adequate rate; Percentage of CC with adequate release; Percentage of CC with adequate hand position.Ventilations (V): Number of total V; Mean pause time for V in s; Number of effective V; Number of V with adequate volume; Number of V with excessive volume; Number of V with insufficient volume; Mean volume in ml.®  manikin was used, with Laerdal Resusci Anne Wireless software  version 2.0.0.14. The configuration parameters were: tidal volume 6–10 mL/kg (35–55 mL) .Quality parameters were evaluated and disaggregated in CC quality, V quality, and CPR quality. Each variable was expressed as a percentage; variable calculations were based on the following formulas published in previous studies ,15:CC quFor the physiological analysis, the percentage of maximum Heart Rate during CPR (%HRmax) was measured. The HR was measured with a Polar Team sensor . The Rating of Perceived Exertion (RPE) was measured on a 0–10 scale ,15,16,17The sample size was based on an assumed minimum effect size of 0.50, an alpha error probability of 0.05, and a statistical power of 0.95. These assumptions required a sample size of 12 study participants as computed by G*Power 3.1.9.2 software . A final sample size of 13 participants was selected to perform this study.All statistical analyses were performed with SPSS version 20 for Windows . The variables were described using measures of central tendency (median and mean), dispersion [interquartile range (IQR) and standard deviation (SD)], or 95% confidence intervals. For comparisons, the Shapiro–Wilk test was used first to check the normality of the data. For parametric variables, a repeated measures ANOVA test with Bonferroni correction was used, and for non-parametric variables comparisons were analyzed using the Friedman test with the Bonferroni correction and the Wilcoxon signed- rank test. For effect size, both Cohen’s d test and Rosenthal’s r test were used. A significance value of 0.05 was set for all tests.A total number of 39 tests were performed by 13 lifeguards (85% were men). The median age of the participants was 26 years (IQR: 23–29 years), the median height was 173 cm (IQR: 166–180 cm), and the median weight was 81 kg (IQR: 64–88 kg).p = 0.032; ES = 0.50) and W-CPR . Lower values were also seen in CC with adequate hand position in R-CPR  when compared to S-CPR  than the S-CPR test . The mean volume was significantly higher in the S-CPR test  than in the W-CPR  and R-CPR  tests. Differences were observed in the rest of the V variables.In the CC analysis, significantly lower results were found in CC with adequate release when comparing R-CPR  to S-CPR . The W-CPR presented a CC Quality of 60 ± 16%; 95% CI: 50–69%. Regarding the Quality of V, no significant differences were observed between the three tests; the medians of quality in values ranged between 43% and 48%. Nor were any differences observed in the Quality of CPR, the best value being obtained in S-CPR  followed by W-CPR  and R-CPR .In relation to the Quality of CC, significantly lower values were observed in R-CPR  than in S-CPR . This represents an average speed of 11.2 km/h (IQR: 10.7–11.8 km/h) during R-CPR and 6.0 km/h (IQR: 5.8–7.7 km/h) during W-CPR (p = 0.002). For this, the participants needed to take 334 steps (IQR: 333–357 steps) during R-CPR and 270 steps (IQR: 267–293 steps) during W-CPR (p = 0.002). R-CPR represented a significant physiological increase over W-CPR and S-CPR. Similarly, W-CPR also represented a significant increase over S-CPR . The same occurred when assessing RPE .Physical condition had a median of 7.0 out of 10.0 (IQR: 4.5–7.5). During the 2 min of R-CPR, rescuers covered 374 m (IQR: 358–392 m), significantly more than during W-CPR  R-CPR is possible, although the total quality presents a slight, non-significant decrease, (b) the quality of compressions during R-CPR decreases by 10% compared to S-CPR, but there are no differences with respect to W-CPR, (c) no differences were observed in airway management during the CPR tests.This research provides novel data on R-CPR. To date, no studies have been published that address this issue. The overall quality of the CPR was very similar in all three simulations. Furthermore, the only published study that evaluated W-CPR did not observe significant differences in total quality either, with values very similar to those of our study (S-CPR: 59% and W-CPR: 49%) . In addiHigh-quality chest compression is very important for the survival of patients in cardiac arrest . Our resThe trained rescuers in our research obtained compression quality values of around 50%. Although recent research shows that professional lifeguards can perform compressions with over 80% quality on an infant manikin , Martin A systematic review and meta-analysis found that the rate of chest compressions is associated with survival outcomes . TherefoStudies in adults and animals have observed that incomplete chest recoil negatively affects hemodynamics, since it increases intrathoracic pressure and decreases venous return to the heart ,23,24,25Donoghue et al. showed that the depth of compressions is a deficient skill in the rescue of infant victims . An unexSimilar to our study, Smereka et al. observed that the optimal way to perform infant resuscitation was on the rescuer’s forearm in a static position, although this research focused on newborn CPR simulations . HoweverA surprising and positive result of the current study was that rescuers did not have difficulty performing rescue breaths during CPR on the move, as was the case in the W-CPR investigation performed by trained lay rescuers . HoweverFatigue negatively affects the quality of an activity which requires significant physical effort, such as CPR ,34. In oThis study has several implications for practice and is based on the research team’s experience in a real pediatric emergency situation that gave rise to the research question. In the education and training of first responders, priority should be given to the most common actions related to prevention, but special resuscitation situations should also be considered. Although these are rare, they require specialized preparation and training. Trained lifeguards can be an asset during a pediatric cardiac arrest. According to the results of this research, trained rescuers can perform acceptable pediatric walking and running CPR even under highly fatiguing conditions.The results of this research are not limited to beaches that are difficult to access but can apply to any aquatic areas  where there is no road access for an ambulance or where there are difficulties in accessing specialized help.This research has several limitations that should be mentioned. On the one hand, it was carried out in a simulated environment, so motivational factors and stress are different from real life. On the other hand, varying distances, weather conditions, locations, or types of rescuers, respectively, could result in different CPR outcomes. In addition, alternative CPR times could provide different results; 2-min tests were performed in the current study, and further research is needed on longer duration CPR while walking or running. As with all simulation studies, the results cannot be directly extrapolated to the care provided to real victims. The strengths of this study are the novelty and relevance of the topic, as well as the limited previous evidence in this context.Under simulated CPR conditions, as rescuers transport an infant victim to safety or to seek expert help while walking or running, lifeguards in good physical condition can perform CPR of a similar quality to when the victim is lying down in a fixed position on a hard surface. The components of CPR quality that worsen under moving conditions are chest compression release and hand position.CPR courses should include practice with victim evacuation scenarios. Moreover, during such trainings instructors should reinforce the following aspects of CPR: frequency, chest compression release, and hand position.